Davide Ciardiello

Primary and Acquired Resistance of Colorectal Cancer Cells to Anti-EGFR Antibodies Converge on MEK/ERK Pathway Activation and Can Be Overcome by Combined MEK/EGFR Inhibition

Purpose: The EGFR-independent activation of the RAS/RAF/MEK/MAPK pathway is one of the resistance mechanisms to cetuximab. Experimental Design: We have evaluated, in vitro and in vivo, the effects of BAY 86-9766, a selective MEK1/2 inhibitor, in a panel of human colorectal cancer cell lines with primary or acquired resistance to cetuximab. Results: Among the colorectal cancer cell lines, five with a KRAS mutation (LOVO, HCT116, HCT15, SW620, and SW480) and one with a BRAF mutation (HT29) were resistant to the antiproliferative effects of cetuximab, whereas two cells (GEO and SW48) were highly sensitive. Treatment with BAY 86-9766 determined dose-dependent growth inhibition in all cancer cells, including two human colorectal cancer cells with acquired resistance to cetuximab (GEO-CR and SW48-CR), with the exception of HCT15 cells. Combined treatment with cetuximab and BAY 86-9766 induced a synergistic antiproliferative and apoptotic effects with blockade in the MAPK and AKT pathway in cells with either primary or acquired resistance to cetuximab. The synergistic antiproliferative effects were confirmed using other two selective MEK1/2 inhibitors, selumetinib and pimasertib, in combination with cetuximab. Moreover, inhibition of MEK expression by siRNA restored cetuximab sensitivity in resistant cells. In nude mice bearing established human HCT15, HCT116, SW48-CR, and GEO-CR xenografts, the combined treatment with cetuximab and BAY 86-9766 caused significant tumor growth inhibition and increased mice survival. Conclusion: These results suggest that activation of MEK is involved in both primary and acquired resistance to cetuximab and the inhibition of EGFR and MEK could be a strategy for overcoming anti-EGFR resistance in patients with colorectal cancer. Clin Cancer Res; 20(14); 3775–86. ©2014 AACR.

Implication of the Hedgehog pathway in hepatocellular carcinoma

The prognosis for patients who are diagnosed with advanced stage hepatocellular carcinoma (HCC) is poor because there are few treatment options. Recent research has focused on the identification of novel molecular entities that can be targeted to inhibit oncogenic signals that are involved in the carcinogenesis, proliferation and progression of HCC. Among all of the pathways that are involved in the development of HCC, Hedgehog (HH) signalling has demonstrated a substantial role in hepatocarcinogenesis and HCC progression. HH plays a physiological role in embryogenesis, through the induction of the differentiation of hepatocytes from endodermal progenitors. The re-activation of the HH pathway in chronic damaged liver is a mechanism of fibrotic degeneration and is implicated in various stages of HCC development. HH activation sustains the sub-population of immature liver epithelial cells that are involved in the pathogenesis of cirrhosis and HCC, and HH itself is a mediator of the alcohol-derived malignant transformation of liver cells. High levels of expression of HH protein markers in liver tumour tissues are correlated with aggressive histological and biological features and a poor clinical outcome. In vitro and in vivo inhibition models of the HH pathway confirm that HH is essential in maintaining tumour growth, metastasis and a mesenchymal phenotype.

Maintenance Treatment with Cetuximab and BAY86-9766 Increases Antitumor Efficacy of Irinotecan plus Cetuximab in Human Colorectal Cancer Xenograft Models

Purpose: The use of cetuximab in the treatment of metastatic colorectal cancer is limited by development of resistance. Experimental Design: We have investigated in three models of highly epidermal growth factor receptor (EGFR)–dependent colorectal cancer xenografts, the effect of maintenance therapy with different kinase inhibitors alone or in combination with cetuximab, after cytotoxic treatment induction with irinotecan plus cetuximab. Results: SW48, LIM 1215, and GEO colorectal cancer cell lines were engrafted into nude mice and treated for 3 weeks with irinotecan and/or cetuximab. The combined treatment induced a significant reduction of tumor size. A subsequent experiment was performed in all three xenograft models in which after an induction treatment with irinotecan plus cetuximab, mice were randomly assigned to one of the following treatments: control, cetuximab, regorafenib, a selective PIK3CA inhibitor (PIK3CAi), a selective MEK inhibitor (MEKi), and/or the combination of each inhibitor with cetuximab. The cetuximab plus MEKi treatment determined the best antitumor activity with suppression of tumor growth. This effect was prolonged for 13 to 15 weeks after cessation of therapy and was accompanied by prolonged survival. Antitumor activity was accompanied by inhibition of the MAPK and MEK pathways. Moreover, in the cetuximab plus MEKi-treated SW48 xenograft group, KRAS mutations as a mechanism of acquired resistance were detected in 25% of cases compared with 75% KRAS mutations in the MEKi-treated group. Conclusions: A possible strategy to prevent and/or overcome resistance to anti-EGFR inhibitors in metastatic colorectal cancer is a maintenance therapy with cetuximab plus MEKi after an initial treatment with irinotecan plus cetuximab. Clin Cancer Res; 21(18); 4153–64. ©2015 AACR.

Sequential HER2 blockade as effective therapy in chemorefractory, HER2 gene-amplified, RAS wild-type, metastatic colorectal cancer: learning from a clinical case

Background Constitutive activation of HER2-dependent intracellular signalling by HER2 gene amplification or by HER2 mutations has been demonstrated as a mechanism of primary and secondary cancer resistance to cetuximab or panitumumab in preclinical and clinical models of metastatic colorectal cancer (mCRC). Both HER2 Amplification for Colorectal Cancer Enhanced Stratification (HERACLES) cohort A and My Pathway clinical trials provided clinical evidence that anti-HER2 therapies could be active in these patients. Patient and methods HER2 gene amplification and HER2 protein overexpression analysis were performed in tumour tissue by fluorescence in situ hybridisation and immunohistochemistry. HER2 positivity was defined according to HERACLES CRC-specific HER2 scoring criteria. DNA analysis for multiple assessment of gene mutations or amplifications was carried out with the next-generation sequencing (NGS) Ion AmpliSeq Colon and Lung Cancer Panel and by using a more extensive targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay). Results We report the clinical case of a patient with HER2 gene amplified and RAS/BRAF wild-type mCRC who experienced a long lasting and relevant clinical efficacy from sequential anti-HER2 therapies (trastuzumab plus lapatinib, pertuzumab plus trastuzumab, trastuzumab emtansine, trastuzumab plus capecitabine) achieving a cumulative clinical benefit of 29 months, after failure of the first three lines of standard treatments, which included all the potentially active drugs in mCRC, and which accounted for only 14 months of disease control. HER gene amplification was confirmed by NGS on two different metastatic lesions during the evolution of the disease. Conclusion The clinical case highlights the role of HER2 gene amplification as a key genetic driver of cancer development and progression in mCRC and suggests that sequential HER2 blockade could be a potential therapeutic strategy.

Present and future of metastatic colorectal cancer treatment: A review of new candidate targets

In the last two decades, great efforts have been made in the treatment of metastatic colorectal cancer (mCRC) due to the approval of new target agents for cytotoxic drugs. Unfortunately, a large percentage of patients present with metastasis at the time of diagnosis or relapse after a few months. The complex molecular heterogeneity of this disease is not completely understood; to date, there is a lack of predictive biomarkers that can be used to select subsets of patients who may respond to target drugs. Only the RAS-mutation status is used to predict resistance to anti-epidermal growth factor receptor agents in patients with mCRC. In this review, we describe approved targeted therapies for the management of metastatic mCRC and discuss new candidate targets on the horizon.

Therapeutic efficacy of SYM004, a mixture of two anti-EGFR antibodies in human colorectal cancer with acquired resistance to cetuximab and MET activation

Purpose Cetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit. Experimental Design We tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab. Results SYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug. Conclusion These results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.PURPOSE Cetuximab and panitumumab have an effective therapeutic response in a subset of RAS Wild-Type (WT) metastatic colorectal cancers (mCRCs). Despite molecular-driven selection, all patients do not respond to epidermal growth factor receptor (EGFR) inhibitors and the onset of secondary resistance limits their clinical benefit. EXPERIMENTAL DESIGN We tested, in vitro and in vivo, the effect of SYM004, a 1:1 mixture of two recombinant human-mouse chimeric monoclonal antibodies (mAbs) directed against non-overlapping epitopes of the EGFR, on CRC models with acquired resistance to cetuximab. RESULTS SYM004 showed a potent growth inhibitory effect in CRC cell lines with acquired resistance to cetuximab and MET activation. SYM004 treatment determined a significant induction of apoptosis and a strong inhibition of MET, AKT and MAPK phosphorilation in these resistant models. The data may further suggest SYM004 -driven induced internalization and degradation of the antibody-receptor complex, which prevents cross-interaction between EGFR and MET even in the presence of TGFα. Moreover, in vivo xenograft studies demonstrated that SYM004 has stronger antitumor activity than cetuximab in CRC models. Importantly, in the current work we observed a response to therapy in all cetuximab resistant tumors mice treated with SYM004. More importantly, four out of seven mice continue to respond to SYM004 after 30 weeks of treatment underling the prolonged effect of the drug. CONCLUSION These results suggest that the treatment with SYM004 could be a strategy to overcome acquired resistance to first generation of anti-EGFR therapies in mCRC as a result of MET activation.

Optimization of the Development of Old and New EGFR and MAP Kinase Inhibitors for Colorectal Cancer

The epidermal growth factor receptor (EGFR) and its downstream signaling pathways are crucially important in the biology of colorectal cancer (CRC). In the past few years, EGFR kinase and its major downstream effector, the RAS/RAF/MAPK/ERK pathway, have been attractive targets for development of new therapy for treatment of metastatic CRC (mCRC). Cetuximab and panitumumab, two monoclonal antibodies (mAbs) against the EGFR, are the first targeted agents approved as personalized medicine for treatment of mCRC. Recently, inhibition of MEK1/2 has been seen as a promising approach for blocking MAPK-mediated proliferation signals. Several compounds with highly selective inhibitory effects on MEK1/2 have been developed and have entered clinical trials. New therapeutic agents have also been used to optimize personalized treatment of mCRC. In this context we discuss recent advances in research on EGFR and MAPK inhibitors.

1585PAXL TYROSINE KINASE RECEPTOR AS A KEY REGULATOR OF PROLIFERATION AND SURVIVAL IN COLORECTAL CANCER (CRC)

ABSTRACT Background: Novel therapeutic strategies are evolving in CRC to selectively inhibit oncogenic pathways. In this scenario we identified the tyrosine kinase receptor AXL as a potential target. Methods: We evaluated the expression and activation of AXL kinase and its ligand GAS6 in a panel of CRC cell lines by receptor tyrosine kinase array (RTK), western blot and Real Time PCR. In the AXL expressing cell lines we analyzed cell survival and drug sensitivity (using the AXL inhibitor foretinib) by MTT assay. Finally we generated stable short hairpin RNA (shRNA)-sh-AXL cell line in order to evaluate the effect of AXL specific knockdown in a model of CRC in vitro and to analyze the mechanism underlying AXL activation. Moreover, TGFb expression was measured by Luminex assay. Results: We found AXL expression in LOVO, HCT116, SW480 and SW620 CRC cells whereas the HT29, SW48, SW48-CR (Cetuximab Resistant), Colo205, GEO, GEO-CR (Cetuximab Resistant) and HCT15 CRC cells were AXL-negative. AXL-expressing cell lines were sensitive to foretinib with an IC50 range from 0,5 to 2 mM. AXL inhibition caused a significant phosphorylation decrease of MAPK, AKT and S6 ribosomal protein. This data were further confirmed by stable silencing of AXL with sh-RNA in LOVO cells that resulted in a dramatic reduction of cell proliferation and activation of downstream pathway. Since no GAS6 levels were found by ELISA in the supernatant of cells tested, we tried to investigate the possible mechanism of AXL activation in our model. We found highest levels of TGFb in HCT116 and SW620 cells in medium. The stimulation of AXL expressing cells with a recombinant TGFb, for 20 and 120 minutes, resulted in increase of downstream pathway. Conclusions: This data highlighted AXL as a potential target in CRC. AXL-expressing cells are resistant to anti-EGFR therapies, thus it could be a relevant target for cancer treatment, in fact AXL inhibition by foretinib or sh-RNA resulted in a significant cell proliferation and survival reduction. Finally, we identified the TGFb as a potential activator of AXL-mediated signaling pathway. The in vivo study is currently ongoing on HCT116 ortotopic xenografts in nude mice treated with foretinib and final results will be presented. Disclosure: All authors have declared no conflicts of interest.